The prophage BTP1 is highly conserved among strains of the pathogenic lineage Salmonella Typhimurium ST313. We aimed to analyze the role of BTP1 and the gene bstA(BTP1-encoded) in virulence of S. Typhimurium D23580, the ST313 lineage 2 reference strain. The deletion mutant D23580ΔbstA showed significantly higher replication and survival rates within human-derived THP-1 macrophages than the wild-type (WT) strain, while the mutant isolate ΔBTP1, lacking the full prophage, did not significantly differ from the WT. Interestingly, during mice infection, ΔBTP1 yielded significantly higher counts in all tested organs [spleens, livers and mesenteric lymph nodes (MLN)] than the WT, and organs were significantly enlarged compared to WT-infected animals. D23580ΔbstA significantly outcompeted the WT during competitive infection of mice, and yielded significantly enlarged spleens and MLN compared to WT-infected animals during single strain infection. Moreover, increased cellular infiltration and focal necrosis were observed in the liver samples of mice infected with D23580ΔbstA and ΔBTP1 compared to WT-infected animals. In conclusion, removal of the gene bstA and the prophage BTP1 in S. Typhimurium D23580 led to increased virulence in mice, demonstrating that bstA is an antivirulence gene.